Project Summary/Abstract Proper coordination of tissue growth is of fundamental importance both in normal development and in many disease processes, including cancer. Studies in Drosophila and mammalian systems over the past 20 years or so have identified the Hippo pathway as a central regulator of tissue growth. As with many signaling pathways, Hippo components include transmembrane proteins, a cytoplasmic kinase cascade, and output mediated through nuclear transcription factors. However, regulation of Hippo signaling is unusually complex, involving mechanical tension, apical-basal polarity, feedback loops, and regulated assembly of different pathway components at spatially distinct regions of the cell cortex. In addition, further pathway control is exerted by components of the planar cell polarity mechanism, specifically by output from Fat/Dachsous signaling. Fat and Dachsous are both large transmembrane protocadherins that function to control the abundance and localization of an atypical myosin named Dachs. Dachs does not appear be a functional motor protein, but instead functions at the cell cortex to regulate pathway activity at the level of the kinase cascade. Studying the Hippo pathway, and in particular Fat/Dachsous signaling, offers an extraordinary opportunity to learn how multiple inputs that are sensing distinct environmental cues are coordinated to control the output of an important developmental and disease-related growth control pathway. In this proposal, we describe experiments designed to unravel some of the complexity underlying Fat- Dachsous signaling so that we can elucidate the underlying molecular principles essential for its function. First, we propose the notion of a 'core complex' of proteins that interact with and recruit Dachs to the cell cortex where it functions to promote growth. In the absence of upstream regulation, these proteins are maximally active. We describe experiments designed to better understand how this complex forms and what factors regulate its ability to assemble at the cell cortex. Second, we propose the notion that Fat and Dachsous together function to repress activity of the core complex, and thereby control tissue growth. Based on previously published data and our own unpublished observations, we propose a previously unrecognized role for Dachsous in removing Dachs from the cell cortex via endocytosis, and describe an experimental plan to rigorously test this hypothesis. Together, the results of these studies have the potential to fundamentally change our mechanistic understanding of Fat/Dachsous signaling in the Hippo tissue growth control pathway.